Hybridoma producing a monoclonal antibody specific for an antigen of the stratum corneum of human epidermis

ABSTRACT

The subject invention relates to monoclonal antibodies which react with antigenic determinants which are present of the stratus corneum of human epidermis. Furthermore, the invention also relates to methods of using such monoclonal antibodies in immunoassays. One monoclonal antibody of the present invention which is particularly useful is BC 21.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The subject invention relates to monoclonal antibodies reactive withantigenic determinants present on the stratum corneum of human epidermisand uses thereof.

In particular, the invention relates to a monoclonal antibody, referredto as BC 21, which is useful as reagent in the treatment and diagnosisof dermatological diseases and diseases of the internal epithelia.

2. Background Information

It is known that the epidermis contains a basal membrane on which rest alayer of proliferative basal cells and layers of suprabasal cells,comprising the stratum spinosum and the stratum granulosum. These arelayers of cells undergoing differentiation which become graduallykeratinized and ultimately form, on the skin surface, a corneous layer(stratum corneum) composed of dead, completely keratinized cells, i.e.,corneocytes. Corneocytes are formed mainly from keratin filaments whichare enclosed by a very strong membrane called the corneous sheath. Thiscorneous sheath is formed by precursor proteins linked by covalent bondswhen acted upon by a specific enzyme called transglutaminase.

To study skin diseases, for example, psoriasis, acne, warts, papillomasand cancers, differentiation of the normal cells of the epidermis calledkeratinocytes must be better understood. As defined in the presentinvention, the term "normal" signifies "non-pathological." In order toundertake these studies, various markers of the terminal differentiationof the keratinocytes must be made available. A need exists in the fieldto identify terminal differentiation markers of the epidermis withantibodies specific for these determinants.

The present invention discloses the preparation and characterization ofantibodies capable of recognizing antigenic determinants found onstratum corneum of normal human epidermis origin. Antibodies of thistype are capable of reacting specifically with the stratum corneum on anon-pathological section of human epidermis. These new antibodies arepotentially useful for the treatment and diagnosis of many forms ofkeratinization disorders such as ichthyosis, psoriasis and eczema.

SUMMARY OF THE INVENTION

It is a general object of the present invention to provide antibodiesspecific for antigenic determinants expressed on the stratum corneum anduses thereof.

In particular, the present invention relates to antibodies specific forthe stratum corneum on a section of non-pathological human epidermis.

In another embodiment, the present invention relates to antibodiesspecific for the stratum corneum of human epidermis affected bykeratinization disorders such as ichthyosis or photo-induced cutaneousaging, psoriasis or eczema.

In yet another embodiment, the present invention relates to antibodiescharacterized by their discontinuous labeling of the tritium corneum ofthe epidermis in the intercorneocytic space using electron microscopydetection.

In yet another embodiment, the present invention relates to antibodiesspecific for human keratinized epithelial tissue.

A further embodiment of the present invention relates to a hybridomawhich produces a monoclonal antibody specific for an antigen that ischaracterized by expression on stratum corneum of normal humanepidermis.

The present invention also relates to a monoclonal antibody specific foran antigen determinant having the above properties. The class of themonoclonal antibody is IgM.

The present invention also relates to a hybridoma (strain CIRD BC 21)having the accession number of 89091901 deposited on Sep. 19, 1989 atthe European Collectors of Animal Cell Cultures located at the PHLSCentre for Applied Microbiology & Research--Porton Down, Salisbury,Wilts SP4 0JG, United Kingdom. The monoclonal antibody produced byaccession number 89091901 is CIRD BC 21.

In another embodiment, the present invention relates to methods ofpreparing hybridoma cell lines that produce monoclonal antibodiesspecific for the stratum corneum.

A further embodiment of the present invention relates to the preparationof polyclonal antibodies characterized by their specific binding tostratum corneum.

In yet another embodiment, the present invention relates to a method ofdiagnosing normal or pathological differentiation of cells from theepidermis and internal epithelia in a patient comprising the steps ofremoving a tissue or cell sample from a patient, adding an antibody withthe above identified properties to the sample and visualizing thepresence or absence of the antibody in the sample.

A further embodiment of the present invention relates to a method fordiagnosing keratinization disorders in a patient following the stepsdescribed above.

In another embodiment, the present invention relates to a method oftreating keratinization disorders in patients by administering to thepatient a conjugate comprising antibodies specifically for stratumcorneum covalently linked to colloided vectors such as lyposomes ormicropheres that contain a biologically active substance such asmethotrexate or a diagnostic agent.

In yet another embodiment, the present invention relates to apharmaceutical composition comprising the specific antibody covalentlylinked to colloidal vectors such as liposoms or microspheres in aconcentration sufficient to treat a keratinization disorder.

Various other objects and advantages of the present invention willbecome apparent from the tables and the following description of theinvention.

All publications mentioned herein are incorporated by reference.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to antibodies, specifically monoclonalantibodies, that react with antigenic determinants present on thestratum corneum. The present invention further relates to methodsutilizing the antibodies.

One embodiment of the present invention relates to antibodies, inparticular, those pocessing at least one of the following properties:

they recognize the stratum corneum of the human epidermis affected bykeratinization disorders such as ichthyosis or photo-induced cutaneousaging, these disorders possibly presenting, furthermore, an inflammatorycomponent, as in the case of psoriasis or eczema;

they provide discontinuous labeling of the stratum corneum of theepidermis in the intercorneocytic space of, for example,non-pathological human epidermis in immunolabeling detection methodsusing electron microscopy;

they recognize human keratinized epithelial tissues;

they recognize the stratum corneum of the epidermis of at least onemammal other than man.

The present invention also relates to monoclonal antibodies whichrecognize a specific antigen of the stratum corneum of normal humanepidermis, the antigen being that which is recognized by the BC 21monoclonal antibody. The BC 21 nonclonal antibody which recognizes thespecific antigen of the stratum corneum may be easily selected by simpleroutine experiments, for example, by competition tests.

The present invention also relates to antibodies that may be polyclonal,or monoclonal. These antibodies are markers of the terminaldifferentiation of keratinocytes.

The present invention further relates to the antibodies previouslydescribed that are modified by labeling with a tracer agent, usingmethods of preparation of these labeled antibodies known in the art. Forexample, the tracer may be a radiolabel, fluorescent label, or enzymatictracer. Labeling with a radioactive tracer may be achieved by bondingwith a radioactive isotope such as iodine or indium. Bonding antibodieswith a fluorochrome (e.g., fluorescein or rhodamine isothiocyanate) orwith an enzyme is also conventionally known. The enzyme may, forexample, be peroxidase or an alkaline phosphatase. The enzymaticactivity potentially present on the reagent after completion of the testmay be determined using an appropriate substrate which may be detectedby means of, for example, colorimetry, fluorescence, luminescence andpotentiometric analysis.

The present invention further relates to labeled and unlabeledantibodies such as those described above, attached to a solid substrateallowing their use as immunoadsorbent agents in affinity chromatographymethods or as reagents in analytical methods based on theantigen-antibody reaction (radioimmunological, immunofluorescence, andimmunoenzymatic techniques).

One skilled in the art will appreciate that the solid substrate may beproduced using any solid, biological, or synthetic material possessingadsorbent properties or possessing the capability of fixing a linkingagent. Among the solid materials capable of bonding antibodies byadsorption are, for example, polystyrene, polypropylene and latex. Thesolid materials which may be used to fix the antibodies by covalenceusing a linking agent are made of, for example, dextran, cellulose andtheir amino derivatives such as diethylaminoethyl-cellulose ordiethylaminoethyl-dextran. The solid substrate may exist, for example,in the form of microtitration disks, tubes, strips, balls, or plates.The linking agents utilized to fix the antibodies on the solid supportby covalence are generally known in the art; they are bifunctionalderivatives such as dialdehydes and quinones. One skilled in the artwill also appreciate that the antibodies may be bonded in conventionalways on solid mineral substrates.

The present invention also relates to a method for preparing antibodiessuch as those specified above. In the preparation of both monoclonal andpolyclonal antibodies having one or several of the above-mentionedproperties, a vertebrate animal is immunized using purified corneoussheaths from mammal epidermis. The monoclonal or polyclonal antibodiesare collected, purified and selected out using methods known in the art.The epidermis supplying the immunization agent is preferably anon-pathological, and especially a human epidermis.

Mammals whose epidermal corneous sheaths are used as immunization agentsin the procedure described above can be easily determined by routineexperiments by immunizing animals using purified corneous sheaths takenfrom a mammal, and by determining whether the antibodies thus producedcontain antibodies such as those specified above. Among the mammals,exclusive of man, whose corneous epidermal sheaths can be used forimmunization include, in particular, rats, rabbits and miniature pigs.The vertebrate animal immunized is of a species different from that ofthe mammal from which the immunization agent is taken.

The purified corneous sheaths used for immunization may be prepared fromhuman or animal epidermis or stratum corneum. For this purpose, thesample is treated several times by successive incubations at 90° insolutions of sodium dodecyl sulfate (SDS) containingbeta-mercaptoethanol, followed by several electrodialysis operations.These treatments have the effect of solubilizing and removing thebiological substances (lipids and proteins, especially keratin) whichare not bonded in a covalent manner to the structure of the corneoussheath. The purified corneous sheaths are collected, washed, and placedin suspension in physiological serum.

To prepare polyclonal antibodies, antibodies are collected from theserum of immunized animals and purified by conventional methods, forexample, by affinity chromatography, so as to retain only the antibodieswhich recognize the stratum corneum. The antibodies do not recognize thebasal and suprabasal cells of either the epidermis used in thepreparation of the corneous sheaths employed as immunizing agents, orthe normal human epidermis. In addition, the antibodies selected havethe other above-mentioned properties.

To prepare monoclonal antibodies, conventional techniques are used todraw off the lymphocytes from the immunized animal. Next, a proceduresuch as hybridoma cell fusion is performed by fusing the immunized celllymphocytes cultured in vitro, with, for example, the SP2/0 mousemyeloma cells, by adding polyethylene glycol. The appropriate clones ofhybrid cells which secrete monoclonal antibodies having the propertieslisted above are selected after conventional screening procedures arecarried out. Further separation of the selected antibodies produced inthe culture medium or in ascites are performed by methods known in theart.

The present invention also relates to hybrid cell lines capable ofsecreting these antibodies. In particular, the present invention relatesto the CIRD BC 21 hybrid cell line deposited at the European Collectionof Animal Cell Cultures as No. 89091901, as well as the monoclonalantibodies (BC 21) secreted by this cell line.

The present invention further relates to antibodies attached onto asolid substrate that may be reacted with a radiolabeled, fluorescentlabel, or enzymatic marker. The monoclonal antibodies specified abovemay be used as reagents in immunological techniques based on theantigen-antibody reaction. These techniques, such as direct or indirectimmunofluorescence, immunoenzymatic techniques and radioimmunologicalmethods are well known in the art.

The present invention further relates to antibodies that may be used, inparticular, as biological sensors in techniques which use biologicalprobes to detect molecules carrying the antigenic determinant againstwhich the antibody is directed. The antibodies may serve as reagentsused to study the normal or pathological differentiation of theepidermal cells, especially those of the human epidermis. They can beused, in particular, in human medicine for the diagnosis ofdermatological diseases and diseases of the internal epithelia. Inparticular, the antibodies which recognize the keratinized epithelialtissues can be used to determine whether internal epithelia arekeratinized. The antibodies of the present invention may be usedprincipally in all medical imaging techniques using antibodies.

The present invention also relates to antibodies that may be used asreagents for the quantification of the epithelial stratum corneum inorder to study the variation in thickness of the stratum corneum underthe effect of a medication and verify the efficacy of that medication.The antibodies which specifically recognize the stratum corneum in amammal such as rats, rabbits and miniature pigs have the advantage ofmaking possible the selection of that mammal as a model animal for astudy of this kind.

The present invention further relates to antibodies that may also beused in the preparation of medicines, as drug vectors, especially indrugs used in the treatment of disease states linked to keratinizationdisorders, such as ichthyosis or photo-induced aging, or linked tokeratinization disorders with inflammatory components, as in the case ofpsoriasis or eczema. For example, the antibodies according to theinvention which recognize cutaneous tissues affected with psoriasis canbe used as drug vectors in the treatment of that affliction, especiallyantimitotics.

The present invention also relates to antibodies that may be linked by acovalent bond to colloidal vectors such as liposomes (see, for example,Wright and Huang. Advanced Drug Delivery Review 3, 343-389, 1989) orpolymeric microspheres containing a biologically-active substance or adiagnostic agent, in order to be used as immunoreagents or astherapeutic agents. The biologically active substance is, for example,an antimitotic agent such as methotrexate. These liposomes ormicrospheres containing antibodies capable of recognizing cutaneoustissues affected with psoriasis may be used, for example, as amedication in human medicine for the treatment of this affection. Inthis case, the liposomes or microspheres are applied topically.

The following examples illustrate the invention without limiting it.

EXAMPLES Example 1 Production of Monoclonal Antibodies 1) Preparation ofCorneous Sheaths Used As An Antigen

Human epidermis or stratum corneum is incubated for 10 minutes at 90° C.in a solution A (2% SDS, 0.1% DTE, 0.01% sodium azide). Duringincubation, the corneum sheath suspension is vigorously stirred threetimes in a vortex. The sample is then centrifuged for 10 minutes at 4000g and the sediment obtained is put back into suspension in solution A.This entire operation is repeated four times.

The corneous sheath sediment obtained after the final centrifugingprocess is placed in suspension in solution B (62.5 mM Tris-HCL pH 6.8,2% SDS, 5% beta-mercaptoethanol, 10% glycerol, 0.025% bromophenol blue).It is then incubated for 10 minutes at 90° C. and transferred to adialysis bag (Spectrapor 6/132542, marketed by Spectrum Industries). Thebag is then immersed in an electrotransfer tank (Transblot, marketed byBiorad) containing a buffer solution C (200 mM glycine, 25 mM Tris, 0.1%SDS). A voltage of 20 V is fed into the tank for 24 hours at 4° C.

The corneous sheaths are then removed and washed by centrifuging insolution C. The residue obtained is once again placed in suspension inbuffer solution B. Electrodialysis is repeated three times. After thefinal electrodialysis, the residue is put back in suspension indistilled water. The suspension is centrifuged for 5 minutes at 4000 g.This operation is repeated three times, then the residue is placed insuspension in physiological serum in a concentration of 1 mg/ml ofprotein, as determined by the quantitative analysis of Lowry et al. (J.Biol. Chem.. 1951, 193, 265-275).

SDS: sodium dodecyl sulfate

DTE: dithioerythritol

2) Immunization

Female Balb/c mice 8 to 11 weeks old are immunized intraperitoneallywith 1 mg of corneous sheaths prepared as described above. Threeidentical procedures are repeated three weeks apart. The spleens of theanimals are removed three days before the last repetition.

3) Culture of SP 2/0 mice myeloma cells

Use is made of a line of SP 2/0 cells (Shulman and Kohler, Nature. 276,269, 1978) which cannot survive in a medium containing azaserine andhypoxanthine (Buttin et al., Cur. Top. Microbiol. Immunol., 81, 27,1978). The cells are cultured in an Eagle medium modified by Dulbecco(DME) containing 4.5 g/l of glucose, 1.2 g/l of sodium bicarbonate, 1 mMof sodium pyruvate, and 2 mM glutamine (i.e., a complete DME medium), towhich is added 10% heated foetal calf serum (10 minutes at 56° C.).After defrosting, the SP 2/0 cells must be cultured at concentrations ofbetween 2×10⁴ and 8×10⁵ /ml until they have reached a doubling time of12 to 15 hours. Freezing of these cells takes place in a concentrationof 5×10⁶ /ml in 95% foetal calf serum and 5% DMSO (dimethyl sulfoxide).

4) Preparation of the SP 2/0 cells

SP 2/0 cells undergoing exponential growth in a concentration of 10⁵ to2×10⁵ cells per ml are centrifuged at 800 revolutions per minutes forfive minutes, and are put back into suspension in DME without serum inthe proportion of 10⁷ cells/ml.

5) Preparation of the splenocytes

Splenocytes from immunized mice are prepared conventionally and placedin suspension in a complete DME medium (Dulbecco's modified essentialmedium) without serum. For counting purposes, 50 μl of the suspensionare drawn off and mixed with 50 μl of 0.2% trypan blue and 400 μl of PBSbuffer (phosphate buffered saline). After 30 seconds, an aliquot isarranged in a Buerker cell. The suspension is centrifuged at 800 rpm for5 minutes and adjusted to 10⁷ viable cells/ml.

6) Fusion (day 0)

The technique used is derived from the "mass fusion" technique of Joy etal. (J. Immunol. 129, 1153, 1982). The myeloma cells and splenocytes aremixed in a 50-ml polypropylene tube in a ratio of 1 myeloma cells to 4splenocytes, and are centrifuged at 800 rpm for 5 minutes. Thesupernatant fluid is pipetted and 1 ml of complete DME without serum isadded to the sediment, which is then put back into suspension (Galfre etal., Nature. 266, 550, 1977). For 10⁷ splenocytes, 100 μl ofpolyethylene glycol 4000 (PEG 4000) diluted to 50% in DME containing 5%DMSO are added to the sediment. The DMSO must be added immediately. Theadded PEG solution is dripped for 1 minute while slowly shaking the tubecontinuously. After 1 minute resting time at the ambient temperature, 1ml of complete DME containing 10% foetal calf serum (FCS) is addedduring 1 minute. The tube is allowed to rest for 1 minute. Next, 15 mlof complete medium containing the foetal calf serum are dripped(approximately 1 ml per minute) while slowly shaking. The suspensionobtained is drawn off using a 25-ml pipette and distributed in a culturedish 150 mm in diameter, which is incubated in an oven at 37° C. for 6hours. The cells are centrifuged at 800 rpm for 5 minutes and put backin selective medium in the proportion of 1 ml of selective medium to 2.5×10⁶ of the original splenocytes. The selective medium has the followingcomposition: complete DME containing 10⁻⁵ M azaserine, 10⁻⁵ Mhypoxanthine, and 2 μl/ml insulin. The suspension is then distributed inculture plates having 24 wells in the proportion of 0.5 ml/well. Underthese conditions, hybridoma clones appear between day 5 and day 7 0.2ml/well of selective medium is added on day 6.

7) Locating hybridomas which produce the sought-for antibodies

When the hybridomas cover one-third of the surface of the wells, theculture supernatant is tested by indirect immunofluorescence on sectionsof human epidermis using the technique described below.

8) Transfer of the selected wells

When the wells are two-thirds confluent, their content is transferred to12-well plates. At preconfluence, the hybridomas are cloned using thetechnique described below. At the same time, they are developed until aconfluent T25 bottle (25 cm²) is obtained. These cells are then frozenin the freezing medium described above.

For cloning, the dilution limit technique is used. In a plate having 96wells, 1 cell/well is seeded. Another plate is also seeded with atheoretical concentration of 0.3 cell/well. The supernatants in thewells are tested by indirect immunofluorescence and the clones whichreact with all or a part of the epidermis are then cultured anddeveloped in selective medium from which azaserine is absent. After theyhave been cultured for one week, the clones are cultured in completeDME. Cloning is repeated once under the same conditions.

Using the techniques described in Example 2, the clones secretingantibodies which recognize normal human epidermis are selected out.

From the clones selected (category A), those which recognizespecifically the stratum corneum of normal human epidermis (sub-categoryB) are chosen.

It has been observed that, out of the clones in category A,approximately 5% at least (generally about 5 to 10%) belong tosub-category B.

One clone (CIRD BC 21) obtained under these conditions has beendeposited with the European Collection of Animal Cell Cultures, underthe number indicated above.

9) Growth of hybridomas in ascites

The growth of the hybridomas obtained in ascitic fluid is carried out byintraperitoneal injection of 10⁷ cells derived from a single clone into8 week-old BALB/c mice which have been previously sensitized byintraperitoneal injection of 0.5 ml of pristane (2,6,10,14-tetramethylpentadecyl hydride).

10) Identification of the class of gammaglobulins secreted

The class of gammaglobulins secreted in the myeloma supernatant has beenestablished by double diffusion in agar using reagents specific for theheavy gammaglobulin chains (Serotec Ltd., Bicester, Great Britain). Thegammaglobulin secreted by the CIRD BC 21 clone is Ig M.

Example 2 Tissular Specificity of the Antibodies Secreted by the CIRD BC21 Line 1) Immunofluorescence

The tissular specificity of the antibodies present in the myelomasupernatants and in the ascitic fluids has been studied using theindirect immunofluorescence technique on sections of approximately 5 μmof various frozen tissues and included in an inclusion medium(Tissue-Tek). The dried slides which hold the tissues are rinsed for 10minutes in a bath of PBS buffer phosphate, then incubated for 30 minutesin a damp atmosphere in the presence of the monoclonal antibodiesobtained. Rinsing takes place for 10 minutes. A second thirty-minuteincubation is performed in the presence of anti-lgM mice antibodiesbonded with FITC (fluorescein isothiocyanate marketed by Cappel). Afterrinsing under the same conditions as those previously specified, thesections are mounted between slides and cover-slips in the mountingmedium (90% glycerol and 10% PBS containing 1% para-phenylene diamine,pH 8). Epithelial tissues from various sources have been used to studythe species specificity of the antibodies. Different human epithelialtissues have been used to study their tissular specificity. Humanpathological tissues have also been used to evaluate the diagnosticvalue of the antibodies. Furthermore, there activity of the antibodieshas been studied using keratinocytes cultured in conventional cultures(Rheinwald and Green Cell. 6 331,1975), in emerged cultures on deaddermis (Regnier et al Front. Matrix. Biol. 9, 4, 1981), or on collagenlattices (Lenoir et al., Dev. Biol. 130, 610, 1988).

2) Electron Immunomicroscopy

Sections of approximately 10 μm of frozen epidermis in an inclusionmedium (Tissue-Tek) are incubated in the presence of monoclonalantibodies, as before. After rinsing, the sections are incubated withthe ABC Vectastain kit (Vector Lab.). Fixation of the tissue is achievedusing the Graham and Karnovski technique (J. Biochem. Cytochem. 14, 291,1966). The superfine sections, which are dehydrated in aqueous ethanolsolutions having various concentrations containing from 70% to 100%alcohol (volume/volume) and then placed in the EPON 812 inclusionmedium, are observed under a JEOL 1200 EX electron transmissionmicroscope.

3) Results

The immunofluorescence results thus obtained are summarized in thefollowing tables:

                  TABLE I                                                         ______________________________________                                        TISSULAR SPECIFICITY IN MAN OF THE ANTIBODY                                   SECRETED BY THE CIRD BC 21                                                    Tissues tested (by indirect immunofluorescence on sections of                 frozen tissues).                                                                     Esoph-                       Ami-                                      Skin   agus    Cornea  Vagina                                                                              Tongue non  Duodenum                             ______________________________________                                        +++*   --      --      --    -/+(**)                                                                              --   -/+(***)                             ______________________________________                                         *Various sites: Breast, stomach, forehead, prepuce. In all cases, the         antibody attaches only on the stratum corneum.                                **The keratinized zones are positive, while the unkeratinized zones are       negative                                                                      ***The epithelial cells are negative, while the mucous cells are positive     +++: high degree of labeling                                                  --: labeling absent                                                      

                  TABLE II                                                        ______________________________________                                        SPECIFICITY OF ANTIBODY SPECIES                                               SECRETED BY CIRD Bc21 LINE                                                    Species tested (by indirect immunofluorescence                                on sections of frozen tissues                                                                              Miniature pig.sup.a                              Mouse.sup.a                                                                           Rat.sup.a   Rabbit.sup.a,b                                                                         (Gottingen race)                                 ______________________________________                                        --      +++         +++      +++                                              ______________________________________                                         .sup.a Epidermis: antibodies attach only to the stratum corneum.              .sup.b Rabbit lip: The outer orthokeratotic zone is positive, while the       internal parakeratotic zone is negative.                                      +++: high degree of labeling                                                  --: labeling absent                                                      

                  TABLE III                                                       ______________________________________                                        CUTANEOUS PATHOLOGY: ABSENCE OR PRESENCE                                      OF REACTIVITY OF THE ANTIBODIES                                               SECRETED BY THE CIRD BC 21 LINE                                                        Common      Spinocellular                                                                            Basocellular                                  Psoriasis                                                                              Ichthyosis  Carcinomas Carcinomas                                    (5).sup.a                                                                              (1)         (4)        (4)                                           ______________________________________                                        +++      +++         --         --                                            ______________________________________                                         .sup.a the number in parentheses indicates the number of subjects studied     +++: high degree of labeling                                                  --: labeling absent                                                      

                  TABLE IV                                                        ______________________________________                                        REACTIVITY OF THE BC 21 ANTIBODIES                                            WITH CULTURED KERATINOCYTES                                                   Conventional Culture                                                                       Culture on Dermis                                                                           Lattice Culture                                    ______________________________________                                        --           +++           +++                                                ______________________________________                                         +++: high degree of labeling                                                  --: labeling absent                                                      

To summarize, in the case of skin that is normal, psoriasic, or strickenwith common ichthyosis, the antibodies secreted by the CIRD BC 21 linereact exclusively with the stratum corneum. However, they do not reactwith the completely differentiated internal zones, or "corneous globes"of the spinocellular carcinomas. The antibodies do not react withstratified unkeratinized epithelia or with single epithelia. However,specific marking of the mucous cells of the duodenum has been observed.The antibodies also react with the stratum corneum of other animalspecies.

The antibodies do not react with conventionally-cultured keratinocytes,but they strongly label the stratum corneum formed when thekeratinocytes are cultured on dead skin or on collagen lattices that areexposed to air. Finally, immunolabelinging electron microscopy on humanepidermis has revealed the presence of a specific discontinuous labellocated in the intercorneocytic space of the stratum corneum.

What is claimed is:
 1. A hybridoma cell line having the accession numberat the European Collectors of Animal Cell Cultures of No.
 89091901. 2. Amonoclonal antibody produced by the hybridoma of claim 1 wherein saidmonoclonal antibody is CIRD BC 21.